b r a z j i n f e c t d i s . 2 0 1 4;1 8(4):441–444
The Brazilian Journal of
INFECTIOUS DISEASES
www.elsevier.com/locate/bjid
Brief communication
RNA interference inhibits herpes simplex virus
type 1 isolated from saliva samples and
mucocutaneous lesions
Amanda Perse da Silva, Juliana Freitas Lopes, Vanessa Salete de Paula ∗
Laboratório de Desenvolvimento Tecnológico em Virologia, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Rio de Janeiro, RJ, Brazil
a r t i c l e
i n f o
a b s t r a c t
Article history:
The aim of this study was to evaluate the use of RNA interference to inhibit herpes simplex
Received 25 November 2013
virus type-1 replication in vitro. For herpes simplex virus type-1 gene silencing, three dif-
Accepted 6 January 2014
ferent small interfering RNAs (siRNAs) targeting the herpes simplex virus type-1 UL39 gene
Available online 15 May 2014
(sequence si-UL 39-1, si-UL 39-2, and si-UL 39-3) were used, which encode the large subunit of ribonucleotide reductase, an essential enzyme for DNA synthesis. Herpes simplex
Keywords:
virus type-1 was isolated from saliva samples and mucocutaneous lesions from infected
Herpes simplex virus type 1
patients. All mucocutaneous lesions’ samples were positive for herpes simplex virus type-1
Real-time PCR
by real-time PCR and by virus isolation; all herpes simplex virus type-1 from saliva samples
siRNA
were positive by real-time PCR and 50% were positive by virus isolation. The levels of herpes simplex virus type-1 DNA remaining after siRNA treatment were assessed by real-time
PCR, whose results demonstrated that the effect of siRNAs on gene expression depends on
siRNA concentration. The three siRNA sequences used were able to inhibit viral replication,
assessed by real-time PCR and plaque assays and among them, the sequence si-UL 39-1
was the most effective. This sequence inhibited 99% of herpes simplex virus type-1 replication. The results demonstrate that silencing herpes simplex virus type-1 UL39 expression
by siRNAs effectively inhibits herpes simplex virus type-1 replication, suggesting that siRNA
based antiviral strategy may be a potential therapeutic alternative.
© 2014 Elsevier Editora Ltda. All rights reserved.
Herpes simplex virus type 1 (HSV-1) is a member of the Herpesviridae family and is characterized by its ability to establish
latency after primary infection and subsequently reactivated.1
Herpes simplex virus (HSV) is an enveloped, double-stranded
(ds) DNA virus. The HSV-1 genome consists of 152 kb of linear dsDNA arranged as long and short unique segments (UL
and US) flanked by inverted repeated sequences (TRL/IRL and
∗
IRS/TRS, respectively).2 Worldwide prevalence of HSV ranges
from 65% to 90%. HSV-1 gives rise to a spectrum of clinical
manifestations and can still be a major cause of morbidity
and mortality.3 HSV-1 is the causative agent of encephalitis, corneal blindness, and several peripheral nervous system
disorders.4 Beyond the neonatal period, most childhood
herpes simplex virus infections are caused by HSV-1.
Corresponding author at: Laboratório de Desenvolvimento Tecnológico em Virologia, Pavilhão Helio e Peggy Pereira, Instituto Oswaldo
Cruz – Fiocruz, Avenida Brasil, 4365, Manguinhos, 21040-360, Rio de Janeiro, RJ, Brazil.
E-mail address: vdepaula@ioc.fiocruz.br (V.S. de Paula).
http://dx.doi.org/10.1016/j.bjid.2014.01.011
1413-8670/© 2014 Elsevier Editora Ltda. All rights reserved.
442
b r a z j i n f e c t d i s . 2 0 1 4;1 8(4):441–444
The seroprevalence of HSV-1 antibodies increases with age,
reaching 20% by the age of five years. No increase occurs until
20–40 years of age, when 40–60% of individuals are HSV-1
seropositive. Mortality associated with herpes simplex virus
is primarily related to perinatal infection, encephalitis, and
infection in individuals who are immunocompromised.4,5
Recently, RNA interference (RNAi) has emerged as a
new therapeutic strategy against viral infection.6 RNAi
is now widely used to knockdown gene expression, in
a sequence-specific manner.6 It can inhibit the expression of crucial viral proteins by targeting viral mRNAs for
degradation instead of the proteins they encode.7 RNAi is
mediated by 21–25 nucleotide double-stranded small interfering RNA (siRNA) molecules. siRNAs are incorporated into
the RNA-induced silencing complex (RISC), which mediates
mRNA sequence-specific binding and cleavage.8 In particular, siRNAs, processed from double-stranded (ds) RNA
precursors by the type III endoribonuclease Dicer, mediate post-transcriptional gene silencing (PTGS).9 Some studies
confirm that siRNA-directed transcriptional gene silencing
is conserved in mammalian cells.10 Small RNAs may guide
mammalian transcriptional silencing in many different biological contexts.10 HSV-1 encodes its own ribonucleotide
reductase (RR), which reduces ribonucleoside diphosphates to
the corresponding deoxyribonucleotides and is essential for
replication. The HSV-1 RR is formed by a large subunit designated ICP6, encoded by the UL39 gene, and a small subunit
which is encoded by the UL-40 gene. The HSV-1 cannot utilize
cellular RR and therefore is dependent upon its own reductase for replication.11 siRNA-based antiviral therapy may be
a potential effective therapeutic alternative for patients with
acyclovir-resistant HSV strains.
This study evaluated the effects of siRNAs targeting the
HSV-1 UL39 gene on the replication of HSV-1 isolated from
mucocutaneous lesions and saliva samples. Infected patients
with blisters and sores characteristic of herpes skin disease who had HSV-1 DNA detected were included in this
study. For this purpose, samples were collected between 2009
and 2010 after obtaining informed consent statement from
each individual. This study was approved by Ethics Committee of Oswaldo Cruz Foundation (protocol number: 544/09).
The saliva samples were collected using ChemBio (Medford,
New York) and samples from mucocutaneous lesions were
collected using Salivette (Sarsdedt, Germany) devices, respectively. The detection of HSV-1 was confirmed by virus isolation
in cell culture and real-time PCR. In brief, samples from mucocutaneous lesions or saliva samples were suspended in 3 mL
of medium 199 (Sigma) containing antibiotics and antifungical (2.5 ␮g/mL of each one respectively). Vero cell cultures were
inoculated with 300 ␮L of solution containing mucocutaneous
or saliva samples. The HSV-1 strain KOS was used as control of infection.12 During 15 days cell cultures were observed
for viral cytopathic effect typical for HSV infection. HSV
DNA was extracted from clinical specimens using commercial
kits (Qiagen, Valencia, CA, USA) following the manufacturer’s
instructions. HSV-1 specific PCR analysis was conducted with a
SYBR Green real-time PCR assay according to manufacturer’s
instructions. The real-time quantitative PCR was performed
with oligonucleotide primer pairs specific for the coding region
of the glycoprotein D (gD) of HSV-1, as reported previously.13
The primers used were HSV-FP (5 -CGGCCGTGTGACACTATCG3 ) and HSV-RP (5 -CTCGTAAAATGGCCCCTCC-3 ).13 A standard
curve was prepared by serial dilution (101 –107 ) from DNA
extracted from KOS strain (108 copies/mL).
After confirming HSV1 infection, the replication of HSV-1
was inhibited using three siRNA molecules against the UL39 gene from HSV-1 (si-UL 39-1, si-UL 39-2, si-UL39-3).14 One
sequence not targeting any known gene was used as a negative control of siRNA (Applied Biosystems, Foster City, CA,
USA). Vero cells were grown in 6-well plates to 80%–90% confluence and then transfected with specific or control siRNA
using the commercial kit siPORTTM Amine (Ambion/Applied
Biosystems, Foster city, CA, USA). After four hours, the cells
were infected with HSV-1 from the infected patients (25
PFU/mL). At 48–72 h post-infection, plates were fixed with
10% paraformaldehyde for two minutes and then stained
with 1% crystal violet for 30 minutes to count the number of plaques per well. The effects of siRNA in infected
cells were also measured by quantification of HSV DNA.
After 48 h, the DNA from the infected cells was extracted
using a commercial kit (Qiagen) following the manufacturer’s
instructions. Real-time PCR relative quantitative reactions
were performed using SYBR Green real-time PCR Master Mix
(Roche, New Jersey, USA) and 18S RNA was used as the endogenous control. The statistical analysis was performed using
the programs “Graph Pad Prism” 5.0 and Excel. The data
were reported as mean ± standard deviation (SD) and the levels of significance were evaluated using the Student’s t-test
and ANOVA. Differences were considered significant when
p < 0.05.
All samples of mucocutaneous lesions were positive for
HSV-1 by real time-PCR and by virus isolation. The viral load
in mucocutaneous lesions samples ranged from 3.85 × 103 to
9.78 × 104 copies/mL. The saliva samples were all positive for
HSV-1 by real-time PCR, and only 50% of samples were positive by viral isolation. The virus load in saliva samples ranged
from 2.44 × 103 to 1.54 × 104 copies/mL. Previous studies have
shown that the isolation of HSV DNA from saliva, using the
method of viral isolation, is hampered by the presence of substances with anti-HSV activity in saliva.15,16 Using the highly
sensitive technique of real-time PCR, we detected the HSV-1
even in those samples with low viral load. The concentration
of siRNA is important to suppress virus replication. Aiming
to evaluate the best concentration of siRNA required to suppress HSV replication, cells were transfected with siRNAs in
different concentrations (final concentration 3 nM, 6 nM, 9 nM,
12 nM, 15 nM, 18 nM, 21 nM). In this experiment two controls
were used: (1) cells infected with HSV-1 that were transfected
with non-specific siRNA (NC) and cells infected with HSV-1
and not transfected (CIN).
The results demonstrated that these siRNAs could potently
inhibit HSV-1 replication in vitro and was observed that the
concentration of UL39 specific siRNAs to achieve the highest
inhibition of HSV transcription was 6 nM (Fig. 1). This concentration is considerably less than those used in other studies
of gene silencing in mammalian cells, which has typically
ranged from 20 to 200 nM.17–19 However, low concentrations
around 10 nM have also been shown to be sufficient for an
effective silencing of genes.20,21 Following the establishment
of the concentration of siRNA that had the highest inhibitory
Percentage of HSV-1 DNA (%)
b r a z j i n f e c t d i s . 2 0 1 4;1 8(4):441–444
100
si UL 39-1
si UL 39-2
si UL 39-3
80
60
40
20
0
3nM 6nM 9nM 12nM 15nM 18nM 21nM CIN
NC
Sequence
Fig. 1 – Effect of different siRNAs on the expression levels of
HSV-1 DNA. The expression level of HSV-1 DNA in cells
treated with different concentrations (3–21 nM) of siRNAs
(si-UL-39 1, si-UL-39 2, si-UL39 3) was determined by
real-time quantitative PCR. The result was normalized to
the housekeeping gene 18S RNA. NC – cells transfected
with non-specific siRNA (negative control). CIN cells
infected with HSV-1 but not transfected.
effect on UL-39 transcription, a sample from mucocutaneous
lesions with the highest viral load (9.78 × 104 copies/mL) was
used to evaluate the efficiency of gene silencing. The concentration of 6 nM of siRNAs was used for transfection and gene
silencing was evaluated 48 hours post-infection. Analysis by
real-time PCR showed that 99, 90 and 88.5% of gene silencing were achieved upon transfection with siRNAs targeting
si-UL 39-1, si-UL39-2, and si-UL 39-3 sequences, respectively
(Fig. 2). In our study, the siRNA specific to the UL-39-1 sequence
showed the highest level of silencing. By plaque assays from
63 to 69% inhibition of viral plaque formation upon silencing the UL-39 gene. Previous study demonstrated that the
inhibition rates of siRNA1 and siRNA2 on HSV-1 plaque formation were 35.51 and 51.62%.14 The differences in the level
of silencing determined by these siRNA sequences may be
due to the concentration of siRNA, conditions of transfection,
viral strains, type of cell used and differences in the thermodynamic properties of the siRNAs. Currently, there has been
increasing number of studies exploring the potential for RNAi
Percentage of HSV-1 DNA (%)
100
80
60
40
20
IN
C
C
N
3
si
U
L3
9-
9L3
U
si
si
U
L3
9-
1
2
0
Fig. 2 – Inhibition of HSV-1 replication in an infected
patient. The inhibition of UL-39 gene was performed using
siRNAs (si-UL-39 1, si-UL-39 2, si-UL39 3). NC, negative
control; CIN, cells infected with HSV-1 but not transfected.
443
approaches to HSV-1. The siRNAi used in this study silenced
specifically the HSV-1 UL39 gene, which encodes the large subunit of ribonucleotide reductase, ICP6.14,22 RNAi has also been
reported to inhibit HSV-1 replication by using siRNAs targeting glycoprotein E that plays key role in cell-to-cell spread and
virus-induced cell fusion20 ; DNA polymerase gene and VP16
play vital roles in initiation of viral gene expression and viral
proliferation22 and ICP4 is a major regulatory gene required
for efficient transcription of early and late viral genes making it essential for lytic infection.19 These studies that applied
RNAi to interfere HSV-1 infection suggested that these small
sequences might have the potential for effective therapeutic
alternative in patients with HSV-1 infection. One important
aspect of using siRNA activity against DNA viruses is the need
to show the inhibition of viral DNA replication, and when
the amounts of viral DNA were quantified almost no viral
DNA could be detected, which demonstrated the inhibition
of genome replication. Herein, the siRNAi were effective to
inhibit replication of HSV-1 in a strain adapted in cell culture (KOS) and also in wild-type virus isolated directly from
an infected patient with high viral load. The inhibitory effects
were related to the concentration of siRNAi transfected, thus
determining the right concentration siRNAi can improve the
inhibition of virus replication.
Conflicts of interest
The authors declare no conflicts of interest.
Acknowledgements
National Council of Technological and Scientific Development
(CNPq), 478979/2009-6 and Fundação de Amparo à Pesquisa
do Estado do Rio de Janeiro (FAPERJ), E-26/111.561/2010 were
acknowledged for financial support.
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